Focused mutagenesis

In focused mutagenesis experiments, the challenge is to identify the residues where mutagenesis is likely to be beneficial. Indeed, many successful directed evolution experiments show that mutations occur in regions that would be hard or impossible to predict (and difficull to explain that they do), even when a high-resolution structure and much information about the enzyme is available 14, 98-1001. One possibility is to make use of knowledge gained from early rounds of random point... 

The nonstructural region of the precursor, harboring the viral replication machinery, is cut into its mature components in a maturation reaction in which two viral proteases (NS2-pro and NS3/4A-pro) cooperate. Site-directed mutagenesis of an other wise infectious cDNA has shown that both HCV-encoded proteases are necessary for viral infectivity, but most of the attention has so far been focused on one of them a member of the serine protease family (EC 3.4.21) located in the N-terminal region of the viral NS3 protein. 

Systematic experimental and theoretical studies of this kind are helpful in performing in vitro evolution of enantioselectivity. Nevertheless, several questions are not fully answered. Are remote mutations more important than those close to the active site, or is the opposite true Is it more effective to allow randomization all over the enzyme rather than focusing on the region around the active site (or vice versa) To be sure, when applying epPCR or any other mutagenesis method that more or less... 

A variety of approaches exist for stabilizing proteins, for example, chemical modification, immobilization, and site-directed mutagenesis [95,96], but these techniques are not within the scope of this chapter. The focus here will be on stabilization of proteins via formulation development. The principal formulation strategy is to stabilize the protein using clinically acceptable additives (excipients) or through the use of suitable pharmaceutical-processing technologies. 

QSAR models addressing five endpoints relevant for REACH legislation have been developed by the European funded CAESAR research project [56]. These models are focused on BCF in fish, mutagenesis, carcinogenesis, developmental toxicity, and skin sensitization. The developed models have been implemented into a Java-based applet available through the Internet. 

In this chapter I will review some aspects of mutagenesis mechanisms and the mutational consequences of DNA damage generated by ben-zo[a]pyrene. The focus will be on knowledge derived from investigations involving the bacterium Escherichia coli. 

Recent structural studies and electron transfer kinetic experiments focus on structures in which a site-specific covalent crosslink between cytochrome c and cytochrome c peroxidase subunits exists. One of these used site-directed mutagenesis to form a disulfide bond between a V197C mutant CcP and an A81C... 

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